[unreadable] The goal of this R21 application is to demonstrate the feasibility of a global approach to the generation of disease specific monoclonal antibodies (mAbs) to low level proteins for the discovery and validation of biomarkers to cancer. The technology will utilize a workflow that is essentially the reverse of that followed today where antigens are first identified and then antibodies generated for potential biomarkers. In the present application, depleted plasma from lung cancer patients will be used to immunize mice, followed by fusion of the mouse spleens with immortalized cells. The pretreatment will involve a multistep depletion scheme to remove major proteins, leaving a remainder of 1 percent by weight of total protein, almost all of which will be glycosylated species from the use of multilectin affinity chromatography (MLAC) as one of the major depletion steps. More than 1000 hybridomas will be created, and the mAbs will be screened for discriminating antibodies to a pooled lung cancer plasma relative to a pooled controlled plasma, revealing at least 30 discriminating mAbs that are potentially disease specific. To demonstrate that the mAbs are to low level proteins, the antigens for at least 20 mAbs will be determined by affinity isolation and mass spectrometry and the level of the protein in blood estimated or determined. The platform represents a method for biomarker discovery and validation which will possess more than 10-fold greater sensitivity, as well as being 10-fold less expensive and many-fold higher throughput than present day LC/MS approaches. [unreadable] Once feasibility has been demonstrated, the methodology will then be ready for implementation on individual patient samples. Important to the success of this project is the collaboration of the Barnett Institute, a center with expertise in proteomics, with BioSystems International, a company with expertise in immunology and screening. [unreadable] [unreadable] A critical need today is the development of technology that can rapidly and cost effectively deliver disease specific antibodies for low level proteins in blood and that can be used to discover and validate biomarkers for the early detection of cancer in blood. The goal of this program is to meet this need, and the reagents generated should be widely utilized for diagnostic purposes. [unreadable] [unreadable] [unreadable]